Methods for making saccharide-protein glycoconjugates

ABSTRACT

The invention provides a process for the reductive amination of a carbonyl group at the reducing terminus of a polysaccharide, wherein the reductive amination is carried out at a pH between 4 and 5. The invention also provides a process for preparing a conjugate of a polysaccharide and a carrier molecule, comprising the steps of: (a) coupling the polysaccharide to a linker, to form a polysaccharide-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group with a primary amine group in the carrier molecule, to form a polysaccharide-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage. The invention also provides a process for reducing contamination of a polysaccharide-linker compound with unreacted linker, comprising a step of precipitating unreacted linker under aqueous conditions at a pH of less than 5. The invention also provides polysaccharide-linker-carrier molecule conjugates and intermediate compounds obtained or obtainable by these processes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of co-pending U.S. application Ser.No. 15/150,302 filed on May 9, 2016, which is a Continuation of U.S.application Ser. No. 14/343,351 filed on Jun. 2, 2014 (now U.S. Pat. No.9,358,284), which is a National Stage of PCT/IB2012/054805 filed on Sep.14, 2012, which claims the benefit of U.S. Provisional Application No.61/534,751 filed on Sep. 14, 2011. The entire contents of all of theabove applications are hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “2018-06-28 2801-0271PUS2ST25.txt” created on Jun. 28, 2018 and is 916 bytes in size. Thesequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

This invention is in the field of conjugating saccharides, particularlythe core domain from the lipopolysaccharide of Gram-negative bacteria,to carriers in order to form glycoconjugates. The glycoconjugates areuseful for immunisation.

BACKGROUND ART

Saccharides from bacteria have been used for many years in vaccines. Assaccharides are T-independent antigens, however, they are poorlyimmunogenic. Conjugation to a carrier can convert T-independent antigensinto T-dependent antigens, thereby enhancing memory responses andallowing protective immunity to develop. The most effective saccharidevaccines are therefore based on glycoconjugates, and the prototypeconjugate vaccine was against Haemophilus influenzae type b (‘Hib’)[e.g. see chapter 14 of ref. 64].

Gram-negative bacteria are surrounded by an outer membrane that containslipopolysaccharide. Lipopolysaccharides are a diverse group of moleculesthat act as endotoxins and elicit strong immune responses in mammals.Each lipopolysaccharide comprises three parts: an O-antigen (alsoreferred to as the O-specific polysaccharide or O-polysaccharide), acore domain, and a lipid A domain. Antibodies against the O-antigen froma particular Gram-negative bacterium may confer protection againstinfection by that bacterium. Vaccines have therefore been envisaged thatcontain O-antigens conjugated to carrier proteins. For example,O-antigen-based conjugate vaccines have been proposed for variousSalmonellae (e.g. serovars Salmonella Typhimurium and SalmonellaParatyphi A of Salmonella enterica in refs. 1 and 2); Shigella species[refs. 3, 4, 5, 6, 7, 8 and 9]; and Escherichia coli [refs. 10 and 11].In these vaccines, the O-antigen is linked to the core domain from thefull-length lipopolysaccharide (i.e. the conjugated polysaccharide is alipopolysaccharide without its lipid A domain). The polysaccharide isconjugated to the carrier via its core domain. This core domain mayitself induce protective antibodies, and conjugate vaccines havetherefore been envisaged that contain a core domain that is not linkedto an O-antigen [ref.12].

Various methods are known for the conjugation of core domain-containingpolysaccharides to a carrier protein. Some methods involve randomactivation of the polysaccharide chain (e.g. with cyanogen bromide(CNBr) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP))prior to conjugation via a linker (see, for example, refs. 1 and 2).Other methods are more selective, involving a specific residue on thechain (e.g. the 2-keto-3-deoxyoctanoic acid (KDO) terminus of the coredomain). For example, methods in reference 13 involve reductiveamination between the carbonyl group in the KDO terminus with adipicacid dihydrazide (ADH) linker. Reactions involving reductive aminationare typically very slow, e.g. the 7-day step in ref. 13. Anotherselective method is described in ref. 9, this time involving couplingvia the carboxyl group in KDO using1-ethyl-1-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and ADH. In thesenon-selective and selective methods, subsequent reaction with thecarrier typically takes place using EDAC, which activates carboxylgroups in the protein for reaction with the linker. This EDAC activationcan result in activation of the carboxyl group in the KDO subunit,leading to unwanted side reactions. The EDAC activation can also resultin cross-linking of the carrier protein, because activated carboxylgroups in the protein can react with primary amine groups in the proteininstead of in the linker.

Accordingly, there remains a need for further and better ways ofpreparing conjugates, particularly of the core domain from thelipopolysaccharide of Gram-negative bacteria.

DISCLOSURE OF THE INVENTION

The invention is based on conjugation methods that can be used in placeof the conjugation methods of the prior art. These methods may bequicker to perform than the prior art methods, particularly thosemethods that involve reductive amination. The methods also do notrequire the use of EDAC, which avoids unwanted side reactions. Indeveloping these methods, the inventors have found ways of purifyingintermediate compounds that do not require the use of toxic compounds.Accordingly, the methods may be carried out more safely. The resultantconjugates may have different, preferably improved, immunologicalproperties compared to the conjugates of the prior art.

The invention therefore provides alternative or improved methods forconjugating a polysaccharide to a carrier protein, and conjugatesobtained or obtainable by these methods. The invention also providesintermediate compounds that are useful in the methods of the inventionand methods for preparing these intermediate compounds.

First Aspect of the Invention

In a first aspect, the invention provides a process for preparing aconjugate of a polysaccharide and a carrier molecule, comprising thesteps of: (a) coupling the polysaccharide to a linker, to form apolysaccharide-linker intermediate in which the free terminus of thelinker is an ester group; and (b) reacting the ester group with aprimary amine group in the carrier molecule, to form apolysaccharide-linker-carrier molecule conjugate in which the linker iscoupled to the carrier molecule via an amide linkage. Unlike theconjugation processes used in refs. 1, 2 and 13, the process does notrequire activation of the carrier molecule with EDAC, thereby avoidingunwanted side reactions. The invention also provides the individualsteps (a) and (b) of this process; the conjugate obtained or obtainableby this process; and the polysaccharide-linker intermediate obtained orobtainable by step (a) of this process.

Step (a) of the First Aspect of the Invention

In step (a) of this first aspect of the invention, the polysaccharide iscoupled to a linker to form a polysaccharide-linker intermediate inwhich the free terminus of the linker is an ester group. The linker istherefore one in which at least one terminus is an ester group. Theother terminus is selected so that it can react with the polysaccharideto form the polysaccharide-linker intermediate, as explained below.

In some embodiments of the invention, the coupling in step (a) takesplace directly. In these embodiments, the polysaccharide is typicallycoupled to the linker using a primary amine group in the polysaccharide.In these embodiments, the linker typically has an ester group at bothtermini. This allows the coupling to take place by reacting one of theester groups with the primary amine group in the polysaccharide bynucleophilic acyl substitution. The reaction results in apolysaccharide-linker intermediate in which the polysaccharide iscoupled to the linker via an amide linkage. In these embodiments, thelinker is therefore a bifunctional linker that provides a first estergroup for reacting with the primary amine group in the polysaccharideand a second ester group for reacting with the primary amine group inthe carrier molecule. For example, a bifunctional linker of the formulaX₁-L-X₂ may be used, where X₁ is an ester group that can react with theprimary amine group in the polysaccharide; X₂ is an ester group that canreact with the primary amine group in the carrier molecule; and L is alinking moiety in the linker. Typical L groups are straight chain alkylswith 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀), particularly —(CH₂)₄—. Homobifunctional linkers of the formulaX-L-X are particularly suitable, where the two X groups are the same aseach other and are capable of reacting with both of the primary aminegroups; and where L is a linking moiety in the linker. A typical X groupis N-oxysuccinimide. L typically has formula -L′-L²-L′-, where L′ iscarbonyl. Typical L² groups are straight chain alkyls with 1 to 10carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀),particularly —(CH₂)₄—. A typical linker is thus adipic acidN-hydroxysuccinimide diester (SIDEA), and the inventors have found thiscompound to be particularly suitable as the linker for the invention:

In alternative embodiments of the invention, the coupling in step (a)takes place indirectly, i.e. with an additional linker that is used toderivatise the polysaccharide prior to coupling to the linker.

In a first group of embodiments involving indirect coupling, thepolysaccharide is coupled to the additional linker using a carbonylgroup at the reducing terminus of the polysaccharide. This couplingcomprises two steps: (a₁) reacting the carbonyl group with theadditional linker; and (a₂) reacting the free terminus of the additionallinker with the linker. In these embodiments, the additional linkertypically has a primary amine group at both termini, thereby allowingstep (a₁) to take place by reacting one of the primary amine groups withthe carbonyl group in the polysaccharide by reductive amination. Aprimary amine group is used that is reactive with the carbonyl group inthe polysaccharide. The inventors have found that a hydrazide(especially —C(═O)NHNH₂) or hydroxylamino (—ONH₂) group is suitable. Thesame primary amine group is typically present at both termini of theadditional linker. Preferably, the reductive amination is carried outaccording to the process of the second aspect of the invention, which isdescribed in detail below. The reaction results in apolysaccharide-additional linker intermediate in which thepolysaccharide is coupled to the additional linker via a C—N linkage.

In a second group of embodiments involving indirect coupling, thepolysaccharide is coupled to the additional linker using a differentgroup in the polysaccharide, particularly a carboxyl group. Thiscoupling comprises two steps: (a₁) reacting the group with theadditional linker; and (a₂) reacting the free terminus of the additionallinker with the linker. In these embodiments, the additional linkertypically has a primary amine group at both termini, thereby allowingstep (a₁) to take place by reacting one of the primary amine groups withthe carboxyl group in the polysaccharide by EDAC activation. A primaryamine group is used that is reactive with the EDAC-activated carboxylgroup in the polysaccharide. A hydrazide (especially —C(═O)NHNH₂) groupis suitable. The same primary amine group is typically present at bothtermini of the additional linker. The reaction results in apolysaccharide-additional linker intermediate in which thepolysaccharide is coupled to the additional linker via an amide linkage.

These two groups of embodiments involving indirect coupling result in apolysaccharide-additional linker intermediate after step (a₁). Theinvention provides the polysaccharide-additional linker intermediateobtained or obtainable by these embodiments. The invention also providesindividual steps (a₁) and (a₂) of these embodiments.

The free terminus of the additional linker is typically a primary aminegroup. Step (a₂) takes place using this primary amine group in the sameway that the primary amine group is used in the embodiments involvingdirect coupling described above, with the same linker etc. This secondstep results in a polysaccharide-linker intermediate in which thepolysaccharide is coupled to the linker via the additional linker, whichadditional linker is coupled to the linker via an amide linkage. Theadditional linker in these indirect embodiments is therefore typically abifunctional linker that provides a first primary amine group forreacting with the carbonyl (or carboxyl) group in the polysaccharide anda second primary amine group for reacting with one of the ester groupsin the linker. For example, a bifunctional linker of the formula Y₁-L-Y₂may be used as the additional linker, where Y₁ comprises a primary aminegroup that can react with the carbonyl (or carboxyl) group in thepolysaccharide; Y₂ comprises a primary amine group that can react withone of the ester groups in the linker; and L is a linking moiety in theadditional linker. Typical L groups are straight chain alkyls with 1 to10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀),particularly —(CH₂)₄—. Homobifunctional linkers of the formula Y-L-Y areparticularly suitable as the additional linker, where the two Y groupsare the same as each other and are capable of reacting with both thecarbonyl (or carboxyl) group and the ester group; and where L is alinking moiety in the additional linker. A typical Y group is a —NHNH₂group. L typically has formula -L′-L²-L′-, where L′ is carbonyl. TypicalL² groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. C₁,C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀), particularly —(CH₂)₄—. A typicaladditional linker is thus adipic acid dihydrazide (ADH), and theinventors have found this compound to be particularly suitable as theadditional linker for the invention. However, shorter additional linkersmay be used, and the inventors have found that carbodihydrazine (CDH,i.e. Y-L-Y, wherein Y is —NHNH₂ and L is carbonyl) is also particularlysuitable as the additional linker for the invention.

Step (b) of the First Aspect of the Invention

In step (b), the ester group at the free terminus of the linker in thepolysaccharide-linker intermediate is reacted with a primary amine groupin the carrier molecule. The reaction takes place by nucleophilic acylsubstitution to form a polysaccharide-linker-carrier molecule conjugatein which the linker is coupled to the carrier molecule via an amidelinkage.

Between step (a) and step (b), unreacted linker may be removed from thepolysaccharide-linker intermediate. Preferably, this removal is carriedout by the process of the third aspect of the invention, which isdescribed in detail below.

Conjugates with a polysaccharide:protein ratio (w/w) of between 1:10(i.e. excess protein) and 10:1 (i.e. excess polysaccharide) may beproduced by the processes of the invention, depending on the molecularweights of the polysaccharide and the carrier molecule. For example, theconjugates may have excess saccharide, e.g. ratios of 10:1 to 1:1, withratios greater than 1.5:1 being typical for 0-antigen-core from S.Paratyphi coupled to CRM_(1t). Ratios between 8:1 and 1.5:1 are ofparticular interest, more specifically ratios between 6:1 and 2:1. Incontrast, conjugates made by processes of the prior art tend to haveexcess protein, e.g. between 1:17 and 1:1.4 in reference 13. Theinventors have found that ratios between 6:1 and 2:1, particularlybetween 3:1 and 4:1, are useful for conjugates containing O-antigen-corefrom S. Paratyphi and CRM₁₉₇. These conjugates can be manufacturedefficiently and show similar immunogenicity to conjugates containinghigher amounts of polysaccharide (e.g. ratios between 5:1 and 6:1). Interms of polysaccharide:protein ratio (mol/mol), the processes of theinvention allow more than one polysaccharide chain to be conjugated toeach carrier molecule, so ratios greater than 1:1 are typical.

Compositions may include a small amount of free carrier [14]. When agiven carrier protein is present in both free and conjugated form in acomposition of the invention, the unconjugated form is preferably nomore than 5% of the total amount of the carrier protein in thecomposition as a whole, preferably present at less than 2% by weight,and more preferably present at less than 1% by weight.

After conjugation, free and conjugated polysaccharides can be separated.There are many suitable methods, including hydrophobic interactionchromatography (HIC), tangential flow filtration, size exclusionchromatography etc. [see also refs. 15 & 16, etc.].

The conjugate is preferably soluble in water and/or in a physiologicalbuffer.

Second Aspect of the Invention

In a second aspect, the invention provides a process for the reductiveamination of a carbonyl group at the reducing terminus of apolysaccharide, wherein the reductive amination is carried out at a pHbetween 4 and 5. The reductive amination may be carried out at a pHbetween 4.1. and 4.9, such as between 4.2 and 4.8, for example between4.3 and 4.7, such as a pH between 4.4. and 4.6. The inventors have foundthat this relatively low pH allows the reaction to be carried outquickly (e.g. in 1 hour at 30° C.) compared to the 7 day reaction inref. 13.

Preferably the reductive amination is carried out according to theprocess of the first aspect of the invention, specifically step (a₁) ofthe first group of embodiments involving indirect coupling describedabove. Accordingly, the reductive amination may comprise reacting thecarbonyl group with the additional linker described above. Theadditional linker typically has a primary amine group at both termini,thereby allowing the reductive amination to take place by reacting oneof the primary amine groups with the carbonyl group. A primary aminegroup is used that is reactive with the carbonyl group in thepolysaccharide. The inventors have found that a hydrazide (especially—C(═O)NHNH₂) or hydroxylamino (—ONH₂) group is suitable. The sameprimary amine group is typically present at both termini of theadditional linker. The reaction results in a polysaccharide-additionallinker intermediate in which the polysaccharide is coupled to theadditional linker via a C—N linkage. The invention also provides thepolysaccharide-additional linker intermediate obtained or obtainable bythis process.

The additional linker may in particular be a bifunctional linker of theformula Y₁-L-Y₂, where Y₁ comprises a primary amine group that can reactwith the carbonyl group in the polysaccharide; Y₂ comprises a secondreactive group which can be linked to the carrier molecule, such as forexample a primary amine group or a —SH group; and L is a linking moietyin the additional linker. Typical L groups are straight chain alkylswith 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀), particularly —(CH₂)₄—. Homobifunctional linkers of the formulaY-L-Y are particularly suitable as the additional linker, where the twoY groups are the same as each other and are capable of reacting with thecarbonyl group; and where L is a linking moiety in the additionallinker. A typical X group is a —NHNH₂ group. L typically has formula-L′-L²-L′-, where L′ is carbonyl. Typical L² groups are straight chainalkyls with 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈,C₉, C₁₀), particularly —(CH₂)₄—. A typical additional linker is thusadipic acid dihydrazide (ADH), and the inventors have found thiscompound to be particularly suitable as the additional linker for theinvention. However, shorter additional linkers may be used, and theinventors have found that carbodihydrazine (CDH, i.e. Y-L-Y, wherein Yis —NHNH₂ and L is carbonyl) is also particularly suitable as theadditional linker for the invention.

This process is particularly suitable when the polysaccharide is theO-antigen-core from a lipopolysaccharide, particularly from S. ParatyphiA, S. Typhimurium or S. Enteritidis.

Reductive amination is a standard technique in organic chemistry, andhas been used extensively in the production of conjugates of capsularpolysaccharides for vaccine use, including O-antigen-core [13]. In thesecond aspect of the invention, a carbonyl group in the polysaccharidereacts with a primary amine group, e.g. in the additional linkerdescribed above. This step also takes place in the process of the firstaspect of the invention, specifically step (a₁) of the first group ofembodiments involving indirect coupling described above. The reductiveamination can conveniently be achieved by combining the polysaccharidewith the primary amine group in the presence of an appropriate reducingagent (e.g. cyanoborohydrides, such as sodium cyanoborohydride NaBH₃CN;borane-pyridine; sodium triacetoxyborohydride; borohydride exchangeresin; etc.). The skilled person would be capable of identifyingsuitable conditions for reductive amination. For example, the inventorshave found that treatment of polysaccharide (O-antigen-core from S.Paratyphi, S. Typhimurium or S. Enteritidis) at 40 mg/ml in 100 mMsodium acetate at pH 4.5 with additional linker (ADH or CDH) at a 2:1polysaccharide:additional linker ratio (w/w) and NaBH₃CN at a 2:1polysaccharide:NaBH₃CN ratio is suitable. The reaction is typically leftfor 1 hour at 30° C.

Third Aspect of the Invention

In a third aspect, the invention provides a process for reducingcontamination of a polysaccharide-linker intermediate with unreactedlinker, comprising a step of precipitating unreacted linker underaqueous conditions at a pH of less than 5. This process does not requirethe use of toxic solvents such as dioxane or ethyl acetate. Inparticular, the process allows the decontamination to take place underaqueous conditions while avoiding polysaccharide-linker intermediatedeactivation (e.g. by hydrolysis).

Preferably, the polysaccharide-linker intermediate is the intermediateobtained or obtainable by step (a) of the first aspect of the invention,described above. This process is particularly suitable when the linkeris SIDEA. The polysaccharide may in particular be the O-antigen-corefrom a lipopolysaccharide, particularly from S. Paratyphi A, S.Typhimurium or S. Enteritidis.

Typically, the pH at which precipitation takes place is between 2 and 5.In embodiments of the invention the pH may be between 2-3, 3-4 or 4-5.Any suitable aqueous solution may be used to obtain this pH. Forexample, the inventors have found that addition of an aqueous buffersolution, e.g. aqueous citrate buffer, is suitable. The citrate buffermay for example be 100 mM sodium citrate at pH 3. The volume of addedaqueous solution is selected to ensure that the unreacted linker isinsoluble in the final mixture. For example, the inventors have foundthat a water content (vol/vol) of >60% (e.g. 61, 62, 63, 64, 65, 66, 67,68, 69, 70% etc.) is suitable, particularly for the SIDEA linker. Theseconditions may be obtained, for example, by tripling the total volume ofthe reaction mixture with 100 mM sodium citrate solution at pH 3. Themixture is typically left for about 30 minutes. The precipitate isremoved, e.g. by centrifugation.

The inventors have also found that an aqueous solution of hydrochloricacid (HCl) is suitable to reduce the pH. For example 2× volume of HCl82.5 ppm is added to the reaction mixture to a concentration of HCl of55 ppm in the final mixture, resulting in a pH of 2.3. The solution ismixed at 4° C. for 30 min and the solution recovered by centrifugation.

The polysaccharide-linker intermediate may be recovered from solution byprecipitation with alcohol. In this way, the contamination of thepolysaccharide-linker intermediate with unreacted linker is reduced,e.g. to less than 10% by the number of moles of unreacted linkerrelative to the number of moles of linker in the intermediate.

The alcohol may in particular be ethanol, although other lower alcoholsmay be used (e.g. methanol, propan-1-ol, propan-2-ol, butan-1-ol,butan-2-ol, 2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols etc.). Theethanol is typically added to the solution containing thepolysaccharide-linker intermediate to give a final ethanol concentration(vol/vol) of between 80% and 95%. The optimum final ethanolconcentration may depend on the polysaccharide-linker intermediate. Whenthe polysaccharide is the O-antigen-core from S. Paratyphi A, theinventors have found that a concentration of about 85% is suitable.

The ethanol may be added to the solution containing thepolysaccharide-linker intermediate in pure form or may be added in aform diluted with a miscible solvent (e.g. water). The precipitatedpolysaccharide-linker intermediate is recovered, e.g. by centrifugation.It may be washed before further use.

In one embodiment the intermediate with the SIDEA linker is precipitatedusing 2-propanol (90% final concentration v/v) and the precipitateOAg-SIDEA is washed with absolute ethanol.

Preferably, the recovered polysaccharide-linker intermediate is thenused in step (b) of the first aspect of the invention, described above.The process of this third aspect of the invention may in particular takeplace between steps (a) and (b) of the process of the first aspect ofthe invention.

The Polysaccharide

The invention involves a polysaccharide. Typically, the polysaccharidehas a reducing terminus that is a KDO subunit. The KDO subunit comprisesa carbonyl group. In some embodiments of the first aspect of theinvention (specifically the first group of embodiments involvingindirect coupling described above), the carbonyl group is used forcoupling the polysaccharide to the linker in step (a) of the process. Inthe second aspect of the invention, the carbonyl group takes part in thereductive amination of the process. In both aspects, although it istypical for the polysaccharide to have a reducing terminus that is a KDOsubunit, the skilled person will appreciate that other polysaccharidesmay be used that comprise a carbonyl group at their reducing termini.For example, the inventors have found that the capsular polysaccharideof N. meningitidis serogroup X may be used. Other bacterial capsularpolysaccharides that may be suitable for use in the invention aredescribed below. The KDO subunit also comprises a carboxyl group. Insome embodiments of the first aspect of the invention (specificallywithin the second group of embodiments involving indirect couplingdescribed above), the carboxyl group is used for coupling thepolysaccharide to the linker in step (a) of the process. Again, theskilled person will appreciate that other polysaccharides may be usedthat comprise a carboxyl group. For example, bacterial capsularpolysaccharides that may be suitable for use in the invention aredescribed below

In other embodiments of the first aspect of the invention (specificallythe embodiments involving direct coupling described above), thepolysaccharide is coupled to the linker in step (a) of the process usinga primary amine group in the polysaccharide. In these processes it isnot necessary for the polysaccharide to comprise a carbonyl group at itsreducing terminus or a carboxyl group, although these groups willtypically be present anyway (e.g. when the polysaccharide has a reducingterminus that is a KDO subunit). Again, the skilled person willappreciate that other polysaccharides may be used that comprise aprimary amine group. For example, capsular polysaccharides that may besuitable for use in the invention are described below

The polysaccharide may in particular comprise the core domain from thelipopolysaccharide f a Gram-negative bacterium. The core domain is acomponent of the lipopolysaccharide found in the outer membrane of theGram negative bacterium. The lipopolysaccharide also comprises anO-antigen, which is linked via the core domain to a lipid A domain. Thecore domain has a terminal KDO subunit, which is linked to the lipid Adomain in the native lipopolysaccharide. This KDO subunit may be the KDOsubunit described above that may be at the reducing terminus of apolysaccharide used in the invention. The polysaccharide used in theinvention may therefore comprise a core domain from alipopolysaccharide. In preferred embodiments, this polysaccharide alsocomprises an O-antigen linked to the core domain (referred to herein asan O-antigen-core). The O-antigen-core is in particular the O-antigenand core domain from a specific lipopolysaccharide (i.e. thelipopolysaccharide without its lipid A domain). A typical process forthe purification of these O-antigen-cores is based on the phenol-watermethod of Westphal and Jann, first described in the 1960s [ref. 17],followed by detoxification of the lipopolysaccharide with acetic acid oranhydrous hydrazine. For example, this method was applied to S.Typhimurium in ref. 1; S. Paratyphi A in ref. 2; S. dysentery in ref. 13and E. coli O157 in ref. 10. These methods involve sedimentation of thebacteria; inactivation of the culture by formalin fixation; hot phenolextraction of the lipopolysaccharide; and treatment of the extractedlipopolysaccharide with acetic acid (to remove the lipid A) or anhydroushydrazine (to de-O-acylate the lipid A) prior to purification. Thesemethods may also involve treatment of the disrupted cell mass or theextracted lipopolysaccharide with DNAse, RNAse and proteinase to reduceimpurities.

For performing the reductive amination at the KDO carbonyl groupdescribed herein, lipid A should be removed leaving the KDO group freeto react. For this purpose extraction and purification of polysaccharidecan preferably be performed by acetic acid hydrolosis as decribed in forexample references 1-2-10-13 and 18. However, the invention can beapplied to any suitable O-antigen-core, including O-antigen-coreobtained by different purification methods.

Polysaccharides comprising a core domain are preferred because theyprovide a KDO subunit for coupling the polysaccharide to the linker instep (a) of the first aspect of the invention. In particular, the KDOsubunit comprises a carbonyl group for the first group of embodimentsinvolving indirect coupling described above. The KDO subunit alsocontains a carboxyl group for the second group of embodiments involvingindirect coupling described above. The polysaccharide also provides aKDO subunit for the reductive amination of the second aspect of theinvention. In particular, the KDO subunit comprises a carbonyl group forthe reductive amination described above. In those embodiments of thefirst aspect of the invention that involve coupling of thepolysaccharide to the linker in step (a) of the process using a primaryamine group in the polysaccharide, it is also useful for thepolysaccharide to comprise a core domain from a lipopolysaccharide. Thisis because the core domain typically comprises a primary amine group(e.g. within a phosphoethanolamine group, particularly apyrophosphoethanolamine group as in the S. Paratyphi A core domain). Theuse of such a polysaccharide with a core domain that contains a primaryamine group is therefore preferred for these alternative embodiments ofthe first aspect of the invention. However, the phosphoethanolaminecontent may vary between core domains obtained from different bacteriaor strains of the same bacterium. Accordingly, those embodiments of thefirst aspect of the invention that involve coupling of thepolysaccharide to the linker in step (a) of the process using a KDOsubunit are preferred. Of these embodiments, the processes involvingreductive amination are particularly preferred because the resultant C—Nlinkage may be more stable than the linkage obtained via thephosphoethanolamine group.

Typically, the O-antigen and core domain is from the lipopolysaccharideof a Salmonella bacterium, e.g. from Salmonella serogroups A, B or D,and particularly from Salmonella Paratyphi A. The O-antigens ofSalmonella serogroups A, B and D have been described and are thought toshare a common backbone: →2-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→.The serogroup specificity of Salmonella Paratyphi A is conferred by anα-3,6-dideoxyglucose (α-D-paratose) linked (1→3) to the mannose of thebackbone. The α-L-rhamnose of the backbone is partially O-acetylated atC-3 (ref. 2, FIG. 1a ). The α-D-paratose has also been reported to havevarious degrees of O-acetylation. The O-antigen from S. Paratyphi A issometimes referred to as O:2. The published structure of the O-antigenand core domain from S. Paratyphi A is shown in FIG. 2, including theKDO subunit and primary amine group (within a pyrophosphoethanolaminegroup) in the core domain. The O-antigen in the invention may also befrom S. Typhimurium. The published structure of the repeating unit ofthis O-antigen (sometimes referred to as O:4.5) is shown in FIG. 1b .The O-antigen may also be from S. Enteriditis (O:9, published structureof the repeating unit shown in FIG. 1c ). Naturally-derived O-antigensmay contain structural variations compared to the published structuresfor these O-antigens.

The O-antigen and core domain may also be from Shigella species, e.g.from S. flexneri. Other Shigella species that may provide the O-antigenand core domain used in the invention are S. sonnei, S. dysenteriae andS. boydii [ref. 19]. The O-antigen and core domain may also be from E.coli, e.g. E. coli O157. Other lipopolysaccharide-containingGram-negative bacteria that may provide the O-antigen and core domainused in the invention are Klebsiella pneumonia [ref. 20], Vibriocholerae [ref. 21], Haemophilus influenzae and Neisseria meningitidis[ref. 22].

The polysaccharide may be chemically modified relative to thepolysaccharide as found in nature. For example, the polysaccharide maybe de-O-acetylated (partially or fully), but it is preferred forpolysaccharides comprising O-antigen not to be de-O-acetylated. If ittakes place, then de-acetylation may occur before, during or after otherprocessing steps, but typically occurs before any coupling step. Theeffect of de-acetylation etc. can be assessed by routine assays. Forexample, the relevance of O-acetylation on S. Paratyphi A O-antigen isdiscussed in reference 2. The native O-antigen of S. Paratyphi A is saidin this document to have about 80% O-acetylation. Conjugatedde-O-acetylated O-antigen did not elicit anti-lipopolysaccharideantibodies with bactericidal activity. Accordingly, the S. Paratyphi AO-antigen used in the present invention may have between 0 and 100%O-acetylation, but it is preferred for the O-antigen to be O-acetylated.The level of O-acetylation may depend on the bacterial strain thatprovided the O-antigen. For example, the degree of O-acetylation of theS. Paratyphi A O-antigen may be 10-100%, 20-100%, 30-100%, 40-100%,50-100%, 55-95%, 55-85%, 60-80% or 65-75%. Typically, the degree ofO-acetylation of the S. Paratyphi A O-antigen is 55-85%, particularly65-75%. However, higher degrees of O-acetylation, e.g. 70-100%,particularly 85-95% are also typical in some strains.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (e.g. asdescribed in reference 23), carbon NMR (e.g. as described in reference2), the Hestrin method [24] or HPAEC-CD [25]. O-acetyl groups may beremoved by hydrolysis, for example by treatment with a base such asanhydrous hydrazine [2]. To maintain high levels of O-acetylation on thepolysaccharide, treatments that lead to hydrolysis of the O-acetylgroups are minimised, e.g. treatments at extremes of pH.

Other polysaccharides may be used in the invention. The skilled personwould be capable of identifying suitable polysaccharides based on thepolysaccharide comprising a reactive group used in the process of theinvention (i.e. a carbonyl, carboxyl or primary amino group). Inparticular, bacterial capsular polysaccharides may be used in theinvention. These bacterial capsular polysaccharides may for example befrom N. meningitidis, particularly serogroups A, C, W135 and Y; S.pneumoniae, particularly from serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and33F; S. agalactiae, particularly serotypes Ia, Ib, and III; S. aureus,particularly from S. aureus type 5 and type 8; Haemophilus influenzaeType b; Salmonella enterica Typhi Vi; and Clostridium difficile. Theinvention may also use non-capsular bacterial polysaccharides. Anexemplary non-capsular bacterial polysaccharide is the S. pyogenes GAScarbohydrate (also known as the GAS cell wall polysaccharide, or GASP).The invention may also use non-bacterial polysaccharides. For example,the invention may use glucans, e.g. from fungal cell walls.Representative glucans include laminarin and curdlan.

The polysaccharides may be used in the form of oligosaccharides. Theseare conveniently formed by fragmentation of purified polysaccharide(e.g. by hydrolysis), which will usually be followed by purification ofthe fragments of the desired size.

Polysaccharides can be purified by known techniques. The invention isnot limited to polysaccharides purified from natural sources, however,and the polysaccharides may be obtained by other methods, such as totalor partial synthesis.

The Carrier Molecule

The invention involves the use of carrier molecules, which are typicallyproteins. In general, covalent conjugation of saccharides to carriersenhances the immunogenicity of saccharides as it converts them fromT-independent antigens to T-dependent antigens, thus allowing primingfor immunological memory. Conjugation is particularly useful forpaediatric vaccines [e.g. ref. 26] and is a well known technique [e.g.reviewed in refs. 27 to 35].

Preferred carrier proteins are bacterial toxins, such as diphtheria ortetanus toxins, or toxoids or mutants thereof. These are commonly usedin conjugate vaccines. The CRM₁₉₇ diphtheria toxin mutant isparticularly preferred [36].

Other suitable carrier proteins include the Neisseria meningitidis outermembrane protein complex [37], synthetic peptides [38,39], heat shockproteins [40,41], pertussis proteins [42,43], cytokines [44],lymphokines [44], hormones [44], growth factors [44], artificialproteins comprising multiple human CD4⁺ T cell epitopes from variouspathogen-derived antigens [45] such as N19 [46], protein D fromHaemophilus influenzae [47-49], pneumolysin [50] or its non-toxicderivatives [51], pneumococcal surface protein PspA [52], iron-uptakeproteins [53], toxin A or B from Clostridium difficile [54], recombinantPseudomonas aeruginosa exoprotein A (rEPA) [55], etc.

It is possible to use mixtures of carrier proteins. A single carrierprotein may carry multiple different polysaccharides [56].

Combinations of Conjugates and Other Antigens

As well as providing individual conjugates as described above, theinvention provides a composition comprising a conjugate of the inventionand one or more further antigens. The composition is typically animmunogenic composition.

The further antigen is preferably capsular polysaccharide fromSalmonella Typhi (Vi) or Citrobacter Vi. This capsular saccharide may beconjugated, e.g. to recombinant mutant P. aeruginosa exoprotein A (as inref. 57) or more preferably to CRM₁₉₇ [36, 58, 59]. A preferredVi-CRM₁₉₇ conjugate for use in the present invention is described inreference 60.

The further antigen(s) may comprise further conjugates, either preparedby a process of the invention or by a different process. For example, inone embodiment the present invention provides a composition comprising aconjugate of S. Typhimurium O-antigen-core and a conjugate of S.Enteriditis O-antigen-core, wherein at least one (more typically both)of the conjugates is prepared by a process of the invention. In anotherembodiments, the present invention provides a composition comprising aconjugate of S. Paratyphi A O-antigen-core, a conjugate of S.Typhimurium O-antigen-core and a conjugate of S. EnteriditisO-antigen-core, wherein at least one of the conjugates (e.g. two ormore, typically all three) is prepared by a process of the invention.

In other embodiments, the one or more further antigen(s) are selectedfrom the following

-   -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        61-63; chapters 22 & 23 of ref. 64].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 65, 66; chapter 15 of ref. 64].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 66,67; chapter 16 of ref. 64].    -   an antigen from hepatitis C virus [e.g. 68].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 69 & 70; chapter 21 of ref.        64].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        13 of ref. 64].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 27 of        ref. 64].    -   a saccharide antigen from Haemophilus influenzae B [e.g. chapter        14 of ref. 64]    -   an antigen from N. gonorrhoeae    -   an antigen from Chlamydia pneumoniae [e.g. 71, 72, 73, 74, 75,        76, 77].    -   an antigen from Chlamydia trachomatis [e.g. 78].    -   an antigen from Porphyromonas gingivalis [e.g. 79].    -   polio antigen(s) [e.g. 80, 81; chapter 24 of ref. 64] such as        IPV.    -   rabies antigen(s) [e.g. 82] such as lyophilised inactivated        virus [e.g.83, RabAvert^(M)].    -   measles, mumps and/or rubella antigens [e.g. chapters 19, 20 and        26 of ref. 64].    -   influenza antigen(s) [e.g. chapters 17 & 18 of ref. 64], such as        the haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 84].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 85, 86, 87].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. 88-90].    -   an antigen from S. epidermidis [e.g. type I, II and/or III        capsular polysaccharide obtainable from strains ATCC-31432,        SE-360 and SE-10 as described in refs. 91, 92 and 93].

Where a saccharide or carbohydrate antigen is used, it is typicallyconjugated to a carrier in order to enhance immunogenicity. Conjugationof H. influenzae B and pneumococcal saccharide antigens is well known.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[70]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. These combinations may further comprise an antigenfrom hepatitis B virus and/or a saccharide antigen from H. influenzae B,typically both.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 94to 102]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (usually DNA e.g. in the form of aplasmid) that encodes the protein.

Pharmaceutical Compositions and Methods

The invention provides a pharmaceutical composition comprising (a) aconjugate of the invention and (b) a pharmaceutically acceptablecarrier. Typical ‘pharmaceutically acceptable carriers’ include anycarrier that does not itself induce the production of antibodies harmfulto the individual receiving the composition. Suitable carriers aretypically large, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose [103], trehalose [104], lactose,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. The compositionsmay also contain diluents, such as water, saline, glycerol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 105.

Compositions of the invention may be in aqueous form (i.e. solutions orsuspensions) or in a dried form (e.g. lyophilised). If a dried vaccineis used then it will be reconstituted into a liquid medium prior toinjection. Lyophilisation of conjugate vaccines is known in the art e.g.the Menjugate^(T)m product is presented in lyophilised form, whereasNeisVac-C™ and Meningitec™ are presented in aqueous form. To stabiliseconjugates during lyophilisation, it may be typical to include a sugaralcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose)e.g. at between 1 mg/ml and 30 mg/ml (e.g. about 25 mg/ml) in thecomposition.

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses.

Aqueous compositions of the invention are also suitable forreconstituting other vaccines from a lyophilised form. Where acomposition of the invention is to be used for such extemporaneousreconstitution, the invention provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with thecontents of the syringe being used to reactivate the contents of thevial prior to injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition has a volume of0.5 ml e.g. for intramuscular injection.

The pH of the composition is typically between 6 and 8, e.g. about 7.Stable pH may be maintained by the use of a buffer. If a compositioncomprises an aluminium hydroxide salt, it is typical to use a histidinebuffer [106]. The composition may be sterile and/or pyrogen-free.Compositions of the invention may be isotonic with respect to humans.

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. Immunogenic compositionsused as vaccines comprise an immunologically effective amount ofantigen(s), as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.non-human primate, primate, etc.), the capacity of the individual'simmune system to synthesise antibodies, the degree of protectiondesired, the formulation of the vaccine, the treating doctor'sassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

Within each dose, the quantity of an individual saccharide antigen willgenerally be between 1-50 μg (measured as mass of saccharide) e.g. about1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg.

The compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. The composition may be prepared forpulmonary administration e.g. as an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary. Thecomposition may be prepared for nasal, aural or ocular administratione.g. as spray, drops, gel or powder [e.g. refs 107 & 108]. Success withnasal administration of pneumococcal saccharides [109,110], Hibsaccharides [111], MenC saccharides [112], and mixtures of Hib and MenCsaccharide conjugates [113] has been reported.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention will generally be administered inconjunction with other immunoregulatory agents. In particular,compositions will usually include one or more adjuvants. Such adjuvantsinclude, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 117], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ [chapter 9 of ref. 117]. The degree of crystallinity ofan aluminium hydroxide adjuvant is reflected by the width of thediffraction band at half height (WHH), with poorly-crystalline particlesshowing greater line broadening due to smaller crystallite sizes. Thesurface area increases as WHH increases, and adjuvants with higher WHHvalues have been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g. as seen in transmission electron micrographs)is typical for aluminium hydroxide adjuvants. The pI of aluminiumhydroxide adjuvants is typically about 11 i.e. the adjuvant itself has apositive surface charge at physiological pH. Adsorptive capacities ofbetween 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate (i.e.aluminium hydroxyphosphate sulfate). They may be obtained byprecipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/AI molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. at 200° C.) indicates the presence ofstructural hydroxyls [ch. 9 of ref. 117].

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate-like morphology as seen intransmission electron micrographs). Typical diameters of the particlesare in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigenadsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mgAl⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of theinvention may contain a buffer (e.g. a phosphate or a histidine or aTris buffer), but this is not always necessary. The suspensions arepreferably sterile and pyrogen-free. A suspension may include freeaqueous phosphate ions e.g. present at a concentration between 1.0 and20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.The suspensions may also comprise sodium chloride.

In one embodiment, an adjuvant component includes a mixture of both analuminium hydroxide and an aluminium phosphate. In this case there maybe more aluminium phosphate than hydroxide e.g. a weight ratio of atleast 2:1 e.g. ≥5:1, ≥6:1, ≥7:1, ≥8:1, ≥9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g. ≤5 mg/ml, ≤4 mg/ml, ≤3mg/ml, ≤2 mg/ml, ≤1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of <0.85 mg/dose is preferred.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 [Chapter 10 of ref. 117;see also ref. 114] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

Various suitable oin-in-water emulsions are known, and they typicallyinclude at least one oil and at least one surfactant, with the oil(s)and surfactant(s) being biodegradable (metabolisable) and biocompatible.The oil droplets in the emulsion are generally less than 5 μm indiameter, and advantageously the emulsion comprises oil droplets with asub-micron diameter, with these small sizes being achieved with amicrofluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm are preferred as they can be subjected to filtersterilization.

The invention can be used with oils such as those from an animal (suchas fish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Otherpreferred oils are the tocopherols (see below). Oil in water emulsionscomprising sqlauene are particularly preferred. Mixtures of oils can beused.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brijsurfactants), such as triethyleneglycol monolauryl ether (Brij 30); andsorbitan esters (commonly known as the SPANs), such as sorbitantrioleate (Span 85) and sorbitan monolaurate. Preferred surfactants forincluding in the emulsion are Tween 80 (polyoxyethylene sorbitanmonooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. Asmentioned above, detergents such as Tween 80 may contribute to thethermal stability seen in the examples below.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [114-116],        as described in more detail in Chapter 10 of ref. 117 and        chapter 12 of ref. 118. The MF59 emulsion advantageously        includes citrate ions e.g. 10 mM sodium citrate buffer.    -   An emulsion comprising squalene, an α-tocopherol, and        polysorbate 80. These emulsions may have from 2 to 10% squalene,        from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the        weight ratio of squalene:tocopherol is preferably ≤1 (e.g. 0.90)        as this provides a more stable emulsion. Squalene and Tween 80        may be present volume ratio of about 5:2, or at a weight ratio        of about 11:5. One such emulsion can be made by dissolving Tween        80 in PBS to give a 2% solution, then mixing 90 ml of this        solution with a mixture of (5 g of DL-α-tocopherol and 5 ml        squalene), then microfluidising the mixture. The resulting        emulsion may have submicron oil droplets e.g. with an average        diameter of between 100 and 250 nm, preferably about 180 nm.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [119] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [120]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [121]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. Such emulsions        may be lyophilized.    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 122, preferred phospholipid components        are phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 123, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyidioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [124].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [124].    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [125].

Antigens and adjuvants in a composition will typically be in admixtureat the time of delivery to a patient. The emulsions may be mixed withantigen during manufacture, or extemporaneously, at the time ofdelivery. Thus the adjuvant and antigen may be kept separately in apackaged or distributed vaccine, ready for final formulation at the timeof use. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g. between 5:1 and 1:5) but isgenerally about 1:1.

C. Saponin Formulations [Chapter 22 of Ref 117]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterogeneous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref.126. Saponin formulations may also comprise a sterol, such ascholesterol [127].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs; see chapter 23 ofref. 117; also refs 128 & 129). ISCOMs typically also include aphospholipid such as phosphatidylethanolamine or phosphatidylcholine.Any known saponin can be used in ISCOMs. Preferably, the ISCOM includesone or more of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid ofadditional detergent [130].

A review of the development of saponin based adjuvants can be found inrefs. 131 & 132.

D. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 133. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [133]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [134,135].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 136 & 137.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 138, 139 and 140 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 141-146.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [147]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 148-150. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 151-153.

A particularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC-31™ [154-156]. Thus an adjuvant usedwith the invention may comprise a mixture of (i) an oligonucleotide(e.g. between 15-40 nucleotides) including at least one (and preferablymultiple) Cpl motifs (i.e. a cytosine linked to an inosine to form adinucleotide), and (ii) a polycationic polymer, such as an oligopeptide(e.g. between 5-20 amino acids) including at least one (and preferablymultiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may bea deoxynucleotide comprising 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID NO:1). The polycationic polymer may be a peptide comprising I1-mer aminoacid sequence KLKLLLLLKLK (SEQ ID NO: 2). This combination of SEQ IDNOs: 1 and 2 provides the IC-3 I™ adjuvant.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 157 and as parenteraladjuvants in ref. 158. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 159-166. A useful CT mutant is orCT-E29H [167]. Numerical reference for amino acid substitutions ispreferably based on the alignments of the A and B subunits ofADP-ribosylating toxins set forth in ref. 168, specifically incorporatedherein by reference in its entirety.

E. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [169], etc.) [170], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor. Apreferred immunomodulator is IL-12.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [171] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [172].

G. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes (Chapters 13 & 14 of Ref 117)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 173-175.

I. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 176 and 177.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [178]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [179]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [180]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [181]; (6) SAF,containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) Ribi™ adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 117.

An aluminium hydroxide adjuvant is useful, and antigens are generallyadsorbed to this salt. Oil-in-water emulsions comprising squalene, withsubmicron oil droplets, are also preferred, particularly in the elderly.Useful adjuvant combinations include combinations of Th1 and Th2adjuvants such as CpG & an aluminium salt, or resiquimod & an aluminiumsalt. A combination of an aluminium salt and 3dMPL may be used.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponse.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc. The mammal may also be a farm animal, e.g. a pig or cow. Suchveterinary uses are specifically envisaged. The bird is preferablylivestock, particularly a turkey or other poultry.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a conjugate of the invention inthe manufacture of a medicament for raising an immune response in amammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by a bacterium from which thepolysaccharide is derived.

One way of checking efficacy of therapeutic treatment involvesmonitoring bacterial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the bacterial antigensafter administration of the composition, e.g. using a serum bactericidalantibody assay.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references182-189, etc.

Where the invention concerns an “epitope”, this epitope may be a B-cellepitope and/or a T-cell epitope. Such epitopes can be identifiedempirically (e.g. using PEPSCAN [190,191] or similar methods), or theycan be predicted (e.g. using the Jameson-Wolf antigenic index [192],matrix-based approaches [193], MAPITOPE [194], TEPITOPE [195,196],neural networks [197], OptiMer & EpiMer [198, 199], ADEPT [200], Tsites[201], hydrophilicity [202], antigenic index [203] or the methodsdisclosed in references 204-208, etc.). Epitopes are the parts of anantigen that are recognised by and bind to the antigen binding sites ofantibodies or T-cell receptors, and they may also be referred to as“antigenic determinants”.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. The different steps can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

It will be appreciated that sugar rings can exist in open and closedform and that both forms are encompassed by the invention. Similarly, itwill be appreciated that sugars can exist in pyranose and furanose formsand that both forms are also encompassed. Different anomeric forms ofsugars are also encompassed. KDO may in particular undergorearrangements, particularly under acidic conditions. These derivativeforms of KDO are also encompassed by the invention. For example, whenthe invention involves a KDO subunit (e.g. in the reductive amination ofthe first and second aspects), the subunit may be in one or more ofthese derivative forms.

A primary amine group can be represented by formula NH₂R. The R groupwill typically be electron donating, and includes —NH,_—O,C₁₋₈hydrocarbyl, particularly C₁₋₈alkyl, especially methyl. R is often—CH₃, —C₂H₅ or —C₃H₇. The hydrocarbyl may be substituted with one ormore groups, such as: halogen (e.g. Cl, Br, F, I), trihalomethyl, —NO₂,—CN, —N⁺(C₁₋₆alkyl)₂O⁻, —SO₃H, —SOC₁₋₆alkyl, —SO₂C₁₋₆alkyl,—SO₃C₁₋₆alkyl, —OC(═O)OC₁₋₆alkyl, —C(═O)H, —C(═O)C₁₋₆alkyl, —OC(═O)C₁₋₆alkyl, —N(C₁₋₆alkyl)₂, C₁₋₆alkyl, —N(C₁₋₆alkyl)₂, —C(═O)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)C(═O)O(C₁₋₆alkyl), —N(C₁₋₆alkyl)C(═O)N(C₁₋₆alkyl)₂, —CO₂H,—OC(═O)N(C₁₋₆ alkyl)₂, —N(C₁₋₆alkyl)C(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)C(═S)C₁₋₆alkyl, —N(C₁₋₆alkyl)SO₂N(C₁₋₆alkyl)₂,—CO₂C₁₋₆alkyl, —SO₂N(C₁₋₆alkyl)₂, —C(═O)NH₂, —C(═S)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)SO₂C₁₋₆alkyl, —N(C₁₋₆alkyl)C(═S)N(C₁₋₆alkyl)₂,—NH—C₁₋₆alkyl, —S—C₁₋₆alkyl or —O—C₁₋₆alkyl. The term ‘hydrocarbyl’includes linear, branched or cyclic monovalent groups consisting ofcarbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl andalkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl andaryl groups and combinations thereof, e.g. alkylcycloalkyl,alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl,cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl,arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups. Typicalhydrocarbyl are C₁₋₁₄ hydrocarbyl, more particularly C₁₋₈ hydrocarbyl.In the additional linker used in the invention, the primary amine groupis typically part of a hydrazide (especially —C(═O)NHNH₂) group.

An ester group can be represented by formula —C(═O)OR. The R group willtypically be electron donating, and includes C₁₋₈hydrocarbyl,particularly C₁₋₈alkyl, especially methyl. R is often —CH₃, —C₂H₅ or—C₃H₇. The hydrocarbyl may be substituted with one or more groups, suchas: halogen (e.g. Cl, Br, F, I), trihalomethyl, —NO₂, —CN,—N⁺(C₁₋₆alkyl)₂O⁻, —SO₃H, —SOC₁₋₆alkyl, —SO₂C₁₋₆alkyl, —SO₃C₁₋₆alkyl,—OC(═O)OC₁₋₆alkyl, —C(═O)H, —C(═O)C₁₋₆alkyl, —OC(═O)C₁₋₆ alkyl,—N(C₁₋₆alkyl)₂, C₁₋₆alkyl, —N(C₁₋₆alkyl)₂, —C(═O)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)C(═O)O(C₁₋₆alkyl), —N(C₁₋₆alkyl)C(═O)N(C₁₋₆alkyl)₂, —CO₂H,—OC(═O)N(C₁₋₆ alkyl)₂, —N(C₁₋₆alkyl)C(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)C(═S)C₁₋₆alkyl, —N(C₁₋₆alkyl)SO₂N(C₁₋₆alkyl)₂,—CO₂C₁₋₆alkyl, —SO₂N(C₁₋₆alkyl)₂, —C(═O)NH₂, —C(═S)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)SO₂C₁₋₆alkyl, —N(C₁₋₆alkyl)C(═S)N(C₁₋₆alkyl)₂,—NH—C₁₋₆alkyl, —S—C₁₋₆alkyl or —O—C₁₋₆alkyl. The term ‘hydrocarbyl’includes linear, branched or cyclic monovalent groups consisting ofcarbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl andalkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl andaryl groups and combinations thereof, e.g. alkylcycloalkyl,alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl,cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl,arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups. Typicalhydrocarbyl are C₁₋₁₄ hydrocarbyl, more particularly C₁₋₈ hydrocarbyl.In the linker used in the invention, the ester group is typically aN-hydroxysuccinimide ester group.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates published structures of the repeating units of theO-antigens from a) Salmonella Paratyphi A; b) Salmonella Typhimurium;and c) Salmonella Enteritidis.

FIG. 2 illustrates the structure of the Salmonella Paratyphi A O-antigenrepeating unit and core domain.

FIG. 3 shows the separation of O:2-CRM₁₉₇ from unconjugated componentsby Sephacryl S300HR.

FIG. 4 shows the immunogenicity of different O:2-CRM₁₉₇ conjugates, asmeasured by an ELISA immunoassay for anti-O:2 antibodies.

FIG. 5 is a schematic of a conjugation method of the invention appliedto O-antigen-core.

FIG. 6 illustrates a large scale method for the conjugation of O-antigenfrom Salmonella Paratyphi A to CRM₁₉₇.

FIG. 7 is a schematic of a conjugation method of the invention appliedto the capsular polysaccharide from N. meningitidis serogroup X.

FIG. 8 shows an SDS-PAGE analysis of conjugates of capsularpolysaccharide from N. meningitidis serogroup X and CRM₁₉₇.

FIG. 9 shows the result of Serum Bactericidal Activity (SBA) assaysperformed using day 42 pooled sera from mice immunized with 8 ug ofconjugated or unconjugated O:2 and S. Paratyphi A strain. Data arepresented as percentage of CFU recovered in test sera with active BRC(and in control serum with inactive BRC) compared with CFU present innegative control, per anti-O:2 ELISA antibody unit of each serum pool.

MODES FOR CARRYING OUT THE INVENTION

Summary of Conjugate Production

Different methods were compared for conjugating the O-antigen-core fromS. Paratyphi to CRM₁₉₇. In particular, conjugates obtained by activatingthe O:2 chain randomly, or specifically through the terminal KDOsubunit, were compared. Theoretically, activation of only the KDO wouldnot modify the structure of the repeating saccharides and wouldtherefore result in a better defined and easier to characterizeconjugate.

Two methods were based on prior art methods. In the first of thesemethods, the O-antigen was randomly activated with ADH using CDAP andthen conjugated to CRM₁₉₇ using EDAC. In the second method, the coreregion of the O-antigen was activated with ADH (using the COOH group ofKDO by EDAC) and then conjugated to CRM₁₉₇ using EDAC.

Alternative methods were also tested. A first method involved KDOactivation with ADH through the ketone group by reductive amination,followed by activation with SIDEA, and then conjugation with CRM₁₉₇. Asecond method substituted the ADH with CDH. A third method involvedactivation of O-antigen with SIDEA (without activation of KDO with ADH)using the pyrophosphoethanolamine group on the core region.

Conjugation of O-Antigen-Core to CRM₁₉₇

(Comparative) Method A: Random Activation of the O-Antigen-Core Chainwith ADH by CDAP and Conjugation with CRM₁₉₇ by EDAC

This conjugate was synthesized according to ref. 2, as described indetail below.

O-Antigen-Core Derivatisation with ADH Through CDAP.

30 Cpl of CDAP (100 mg/mL in acetonitrile) was added per ml of 10 mg/mlO-antigen-core in 150 mM NaCl at room temperature. The pH was maintainedat 5.8 to 6.0 for 30 seconds, then 0.2 M TEA was added to increase thepH to 7.0 and the solution mixed at room temperature for 2 minutes. 1 mlof 0.8 M ADH in 0.5 M NaHCO₃ was then added per 10 mg of O-antigen-core.The reaction was carried out for 2 hours at room temperature, and pHmaintained at 8.0 to 8.5 with 0.1 N NaOH. The reaction mixture was thendesalted using a G-25 column against water and the product, designatedas “OAg-(CDAP)ADH”, characterized.

Conjugation of OAg-(CDAP)ADH with CRM₁₉₇.

The OAg-(CDAP)ADH was dissolved in 100 mM MES at pH 5.8. An equal weightof protein was added to give an O-antigen-core:CRM₁₉₇ ratio of 1:1 byweight, with an O-antigen-core concentration of 5 mg/ml. The reactionmixture was placed on ice and EDAC added to a final concentration of 50mM, The reaction was mixed on ice for a further 4 h. The resultingconjugate was designated “OAg-(CDAP)ADH-CRM₁₉₇”.

(Comparative) Method B: Activation of Terminal KDO with ADH by EDAC andConjugation with CRM₉ by EDAC

This conjugate was synthesized according to ref. 9.

O-Antigen-Core_Derivatization with ADH at KDO Through EDAC.

The O-antigen-core was solubilized at 3 mg/ml in 100 mM MES at pH 5.8.ADH was then added at a w/w ratio ADH:O-antigen-core of 1.36, followedby EDAC to a final concentration of 3.7 mM. The reaction was mixed atroom temperature for 4 hours. The reaction mixture was then desaltedusing a G-25 column against water and the product, designated as“OAg-(EDAC)ADH”, characterized.

Conjugation of OAg-(EDAC)ADH with CRM₁₉₇.

The conjugate was prepared according to the method described in Method Aabove for OAg-(CDAP)ADH. The conjugate was designated as“OAg-(EDAC)ADH-CRM₁₉₇”.

Methods C and D: Activation of the Terminal KDO with ADH (Method C) orCDH (Method D) by Reductive Amination and Conjugation with CRM₁₉₇ ViaSIDEA Linker

O-Antigen-Core Derivatization with ADH or CDH at KDO by ReductiveAmination.

After testing different conditions, an optimized protocol for theO-antigen-core derivatization was identified. 0-antigen-core wassolubilized at 40 mg/ml in 100 mM AcONa at pH 4.5. Either ADH or CDH wasadded at a w/w ratio of 1:2 with respect to the O-antigen-core. NaBH₃CNwas then added at a w/w ratio of 1:2 with respect to the O-antigen-core.The solution was mixed at 30° C. for 1 hour. The reaction mixture wasthen desalted using a G-25 column against water and the product,designated as “OAg-ADH” or “OAg-CDH” characterized.

OAg-ADH and OAg-CDH Derivatization with SIDEA.

Either OAg-ADH or OAg-CDH was dissolved in 1:9 (vol/vol) water/DMSO to afinal O-antigen-core concentration of 50 mg/ml. Once the polysaccharidewas in solution, TEA was added to give a molar ratio of TEA/total NH₂groups of 5 and then SIDEA to give a molar ratio of SIDEA/total NH₂groups of 12. The solution was mixed at room temperature for 3 hours. Inpreliminary attempts to purify the SIDEA-derivatised O-antigen-core, theO-antigen-core was precipitated by addition of AcOEt or dioxane (90%volume in the resulting solution) and washing the pellet times with thesame organic solvent (ten times using ⅓ of the volume added for theprecipitation) in order to remove unreacted SIDEA. This process was thenadapted to avoid the use of toxic AcOEt and dioxane reagents. A volumeof 100 mM sodium citrate at pH 3 equal to two times the volume of theSIDEA-derivatised O-antigen-core reaction mixture was added and mixed at4° C. for 30 min. Unreacted SIDEA was precipitated by the low pH andseparated by centrifugation. The SIDEA-derivatised O-antigen-core wasthen recovered from the supernatant by precipitation with absoluteethanol (80% volume in the resulting solution). The pellet was washedwith ethanol twice (using ⅓ of the volume added for the precipitation)and dried. The product, designated as “OAg-ADH-SIDEA” or “OAg-CDH-SIDEA”was characterized.

Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA with CRM₁₉₇.

The OAg-ADH-SIDEA or OAg-CDH-SIDEA was solubilized in NaH₂PO₄ buffer atpH 7.2 and CRM₁₉₇ added to a final protein concentration of 20 mg/ml,final buffer capacity of 100 mM and molar ratio of active ester groupsto CRM₁₉₇ of 30 to 1. The reaction was mixed at room temperature for 2hours.

Method E: Direct Conjugation with CRM₁₉₇ Via SIDEA Linker

The reaction conditions used in the above “OAg-ADH and OAg-CDHderivatization with SIDEA” was also applied to native O-antigen-core(i.e. O-antigen-core that had not previously been derivatized with ADHor CDH). The resulting product was designated as as “OAg-SIDEA”. TheOAg-SIDEA was then conjugated to CRM₁₉₇ by the reaction conditions usedin the above “Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA withCRM₁₉₇”.

Purification of the O-Antigen-Core Conjugates

Conjugates made according to methods A-E above were purified by sizeexclusion chromatography on a 1.6 cm×90 cm S-300 HR column eluted at 0.5ml/min in 50 mM NaH₂PO₄, 0.15 M NaCl at pH 7.2. Different pools werecollected according to free O-antigen-core and free CRM₁₉₇ profiles onthe same column in the same eluting conditions (FIG. 3). The first poolat high molecular weight (corresponding to the purified conjugate) didnot contain free saccharide or free protein.

Analysis of Conjugates

The conjugates were analysed by SDS-PAGE and showed an expected highmolecular weight population smear compared to free CRM₁₉₇. Theconjugates were separated from free O:2 and CRM₁₉₇ by Sephacryl S300HRsize exclusion (1.6×90 cm column; 50 mM NaH₂PO₄, 150 mM NaCl pH 7.2; 0.5mL/min flow). Results are shown in FIG. 3. Purified conjugates were thencharacterized by the phenol sulfuric assay of ref. 209 (total sugar),microBCA (total protein), HPAEC-PAD (sugar composition) and HPLC-SEC(size determination, Kd). Results are shown in Table 1 below.

TABLE 1 Wt/wt Total Presence ratio Kd sugar, of free Protein O:2/ (HPLC-Conjugate μg/mL O:2 μg/mL CRM₁₉₇ SEC) O:2 yes 0.549 CRM₁₉₇ 0.690O:2-(CDAP)ADH- 32.88 no 62.98 0.52 0.439 CRM₁₉₇ pool 1 O:2-(CDAP)ADH-101.09 yes 60.44 1.67 0.534 CRM₁₉₇ pool 2 O:2-CDH-SIDEA- 82.82 no 36.672.26 0.403 CRM₁₉₇ Lot A O:2-CDH-SIDEA- 167.61 no 38.19 4.39 2 peaksCRM₁₉₇ Lot B 0.128 0.413 O:2-ADH-SIDEA- 51.54 no 29.56 1.74 2 peaksCRM₁₉₇ Lot A 0.115 0.388 O:2-ADH-SIDEA- 215.13 no 50.89 4.23 2 peaksCRM₁₉₇ Lot B 0.118 0.421 O:2-(EDAC)ADH- 50.19 yes 31.31 1.60 0.52 CRM₁₉₇ O:2-SIDEA- 108.57 no 52.79 2.06 0.376 CRM₁₉₇, pool 1 O:2-SIDEA-185.31 yes 68.18 2.72 0.457 CRM₁₉₇, pool 2

Imunogenicity Studies

An ELISA immunoassay was used to detect anti-O:2 antibodies elicited byO:2-CRM₁₉₇ immunized mice. For the assay, MaxiSorp microtiter plateswere coated with 15 μg/mL O:2 in a carbonate coating buffer (pH 9.6)overnight at 4° C.

The O:2-CRM₁₉₇ conjugates were compared to unconjugated O:2 antigen.Briefly, groups of female CD1 mice (8 per group at 5 weeks of age) wereinjected subcutaneously with 200 μL of conjugates as set out in Table 2.Mice received immunizations on days 0, 14 and 28. Sera were collectedfrom the mice during the course of the study and tested by the ELISAassay.

TABLE 2 Group Vaccine O:2 antigen dose, μg 1 O:2-(CDAP)ADH-CRM₁₉₇, pool1 1 2 8 3 O:2-(CDAP)ADH-CRM₁₉₇, pool 2 1 4 8 5 O:2-CDH-SIDEA-CRM₁₉₇ 1 68 7 O:2-ADH-SIDEA-CRM₁₉₇ 1 8 8 9 O:2-(EDAC)ADH-CRM₁₉₇ 1 10 8 11Unconjugated O:2 antigen 8

ELISA results through day 42 of the study showed that all theconjugates, except O:2-(EDAC)ADH-CRM₁₉₇ were able to elicit high serumlevels of anti-O:2 IgG antibodies in mice when delivered at the 1 μgdose (FIG. 4). Increases in antibody were observed following the secondvaccination with conjugate, whereas repeated immunization with 8 μg ofunconjugated O:2-antigen did not result in specific IgG antibodies.Delivery of 8 μg doses was no better than 1 μg doses at generating ahumoral immune response in mice (FIG. 4).

Serum Bactericidal Activity

Serum Bactericidal Activity (SBA) assays were performed using day 42pooled sera from mice immunized with 8 ug of conjugated or unconjugatedO:2 and S. Paratyphi A (see Table 2). The result is shown in FIG. 9.Inhibition of S. Paratyphi A growth in vitro correlated with increasinganti-O:2 ELISA units present in the sera pools. The strongest growthinhibition was observed with those conjugates produced using a selectivechemistry; both SIDEA conjugates and O:2-(EDAC)ADH-CRM197 resulted inincreased inhibition compared to O:2-(CDAP)ADH-CRM197, whose O:2 wasrandomly modified prior to conjugation. Even unconjugated O:2, althoughfar from reaching high levels of bacterial growth inhibition due to thelower amount of antibody present in the sera, presented an inhibitionprofile similar to the conjugates prepared with unmodified O:2 chains.No bacterial growth inhibition was detected using control serum in thepresence of iBRC (the same sera in the presence of active BRC is shownas control), indicating a role for complement mediated killing.

Larger-Scale Processing

Based on the immunogenicity study and ease of conjugatecharacterization, the conjugation method based on O:2-activation withADH and then SIDEA and reaction with CRM₁₉₇ (method C, FIG. 5) wasselected for further development. Conjugates made by this method weremore immunogenic than conjugates made by the method B, for example.Although conjugates made by method A gave comparable ELISA titers (FIG.4), they resulted in considerably lower Serum Bactericidal Activity (seeExample and FIG. 9). Furthermore these conjugates had a cross-linkedstructure, with multiple possible points of linkage on thepolysaccharide chain to one or more protein molecules, a withdisadvantages in terms of reproducibility and characterization of theproduct. In contrast, the conjugates of method C contain morepolysaccharide chains per protein molecule, with only one point oflinkage on the polysaccharide chain. The remainder of the polysaccharidechain is left unchanged. Derivatization of the O-antigen-core throughCDAP (method A) can result in crosslinking of the sugar chains and theoverall conjugation method was more complicated. The use of EDAC inmethods A and B can also result in cross-linking of the protein.

A scaled-up process was developed for method C for the production ofgreater amounts of conjugate (FIG. 6). In particular, the step of O:2reductive amination with ADH was optimized to reduce the reaction timeto 1 hour (instead of 7-14 days as previous reported for this kind ofreaction [13]) with good % of O:2 activation (>65%). This step wasscaled to 300 mg of O:2 and repeated several times with reproducibleresults in terms of yield and derivatisation degree. Recovery was >75%after tangential flow filtration, with >70% of O-antigen-core activation(calculated as molar ratio of linked ADH groups per GlcNAc groups on theO-antigen-core) and good purity (ratio of free ADH/linked ADH <1%). Thestability of the O:2-ADH intermediate in aqueous solution at 4° C. wasverified to be >1 month. The inventors envisage replacing the dryingstep by O:2-ADH precipitation in 85% ethanol.

The reaction of O:2-ADH with SIDEA was optimized to avoid the use ofAcOEt during O:2-ADH-SIDEA purification. Removal of free SIDEA byprecipitation at pH 4-5 and then precipitation of O:2-ADH-SIDEA in 85%EtOH showed better precipitate formation that was easier to washcompared with AcOEt. Recoveries >85% were obtained working on a 100 mgscale with activated NH₂ groups >80%.

The inventors have found that in addition to O:2 from S. Paratyphi A theconjugation method based on O-antigen-core activation with ADH and thenSIDEA and reaction with CRM₁₉₇ (method C, FIG. 5), works equally wellfor O-antigen-core from S. Typhimurium and O-antigen-core from S.Enteritidis.

Conjugation of MenX Capsular Polysaccharide to CRM₁₉₇

Method C was also applied to the conjugation of capsular polysaccharidefrom N. meningitidis serogroup X (FIG. 7), resulting in conjugateformation with no free protein in the reaction mixture (FIG. 8).

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

Effect of pH and Temperature on the Reductive Amination of S. Paratyphia O-Antigen-Core with ADH as Linker

Experiments summarized in Table 3 were performed working with S.Paratyphi A O-antigen-core. The effect of different pH and differenttemperatures was evaluated. The best activation was obtained at lower pHand was temperature independent. Reactions were performed in 100 mMbuffer, with O-antigen-core concentration of 20 mg/mL and a ratio of ADHto O-antigen-core and NaBH₃CN to O-antigen-core both of 1.2 to 1 (w/w).ADH and NaBH₃CN were added at the same time and solutions were mixed for1 hour.

TABLE 3 Reductive animation of O:2-KDO with ADH is pH dependent andtemperature independent. Buffer Temperature % activated O:2 Sodiumacetate pH 4.5 30° C. 65.8 Sodium acetate pH 4.5 50° C. 65.4 Sodiumacetate pH 4.5 60° C. 68.3 MES pH 6.0 30° C. 43.9 Phosphate pH 8.0 30°C. 26.2

Derivatization of S. Typhimurium O-Antigen-Core with ADH by ReductiveAmination Comparing Different Reaction Conditions (Table 4).

Reaction conditions described in this application (Table 4, method 1)for performing the reductive amination with ADH were compared with thetraditional method reported in literature [13] (Table 4, method 2),working with S. Typhimurium O-antigen-core (strain D23580). Results aresummarized in Table 5, showing that the process at lower pH is fasterand also more efficient.

TABLE 4 Reaction conditions used for performing reductive animation withADH comparing NVGH procedure with the classical procedure reported inliterature. Temper- OAg ADH NaBH₃CN ature Method (mg/mL) (mg/mL) (mg/mL)Buffer (° C.) 1 40 48 48 AcONa 30 100 mM pH 4.5 2 20 100 100 NaHCO₃ 37100 mM pH 8.3

TABLE 5 Reductive amination of O:4,5-KDO with ADH using method 1 is moreefficient and faster than using the classical method reported inliterature (method 2). Reaction % activated Method time O:4,5 1 3 h 88 23 h 31.6 2 24 h  28.5 2 5 d 28.5

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The invention claimed is:
 1. A polysaccharide-linker intermediate of theformula:

wherein OAg is an O-antigen-core from a lipopolysaccharide which hasbeen coupled using a carbonyl group at the reducing terminus.
 2. Thepolysaccharide-linker intermediate of claim 1, wherein thelipopolysaccharide is from a Salmonella bacterium.
 3. Thepolysaccharide-linker intermediate of claim 2, wherein thelipopolysaccharide is from Salmonella serogroups A, B or D.
 4. Apolysaccharide antigen conjugate prepared by a process comprising thesteps of: (a1) reacting a carbonyl group at a reducing terminus of apolysaccharide with Y₁ of an additional linker of Formula 1, whereinFormula 1 is Y₁-L-Y₂, wherein L is a linking moiety, Y₁ and Y₂ are each—NHNH₂ groups; (a2) reacting Y₂ of the additional linker with a linker,to form a polysaccharide-additional linker-linker compound in which thefree terminus of the linker is an ester group; and (b) reacting theester group of the linker with a primary amine group in a carriermolecule, to form a polysaccharide-additional linker-linker-carriermolecule conjugate in which the linker is coupled to the carriermolecule via an amide linkage.
 5. The polysaccharide antigen conjugateof claim 4, wherein the polysaccharide comprises an O-antigen from alipopolysaccharide.
 6. The polysaccharide antigen conjugate of claim 5,wherein the polysaccharide also comprises a core domain.
 7. Thepolysaccharide antigen conjugate of claim 5, wherein thelipopolysaccharide is from a Gram negative bacterium.
 8. Thepolysaccharide antigen conjugate of claim 6, wherein thelipopolysaccharide is from a Gram negative bacterium.
 9. Thepolysaccharide antigen conjugate of claim 7, wherein thelipopolysaccharide is from a Salmonella bacterium.
 10. Thepolysaccharide antigen conjugate of claim 8, wherein thelipopolysaccharide is from a Salmonella bacterium.
 11. Thepolysaccharide antigen conjugate of claim 4, wherein the carriermolecule is CRM₁₉₇.
 12. The polysaccharide antigen conjugate of claim 4,wherein the additional linker is adipic acid dihydrazide (ADH).
 13. Thepolysaccharide antigen conjugate of claim 4, wherein the polysaccharidecomprises an O-antigen and a core from a lipopolysaccharide from aSalmonella bacterium, wherein the carrier molecule is CRM₁₉₇, whereinthe additional linker is adipic acid dihydrazide (ADH), wherein thelinker is adipic acid N-hydroxysuccinimide diester (SIDEA) and whereinthe additional linker is coupled using a carbonyl group at the reducingterminus of the polysaccharide.
 14. A process for preparing a conjugateof a polysaccharide and a carrier molecule comprising the steps of: (a1)reacting a carbonyl group at a reducing terminus of the polysaccharidewith Y₁ of an additional linker of Formula 1, wherein Formula 1 isY₁-L-Y₂, wherein L is a linking moeity, Y₁ and Y₂ are each —NHNH₂groups; (a2) reacting Y₂ with the linker SIDEA, to form apolysaccharide-additional linker-linker compound in which the freeterminus of the linker is an ester group; and (b) reacting the estergroup of the linker with a primary amine group in a carrier molecule, toform a polysaccharide-additional linker-linker-carrier moleculeconjugate in which the linker is coupled to the carrier molecule via anamide linkage.
 15. The process of claim 14, wherein the polysaccharidecomprises an O-antigen from a lipopolysaccharide.
 16. The process ofclaim 15, wherein the polysaccharide also comprises a core domain. 17.The process of claim 15, wherein the lipopolysaccharide is from a Gramnegative bacterium.
 18. The process of claim 16, wherein thelipopolysaccharide is from a Gram negative bacterium.
 19. The process ofclaim 17, wherein the lipopolysaccharide is from a Salmonella bacterium.20. The process of claim 18, wherein the lipopolysaccharide is from aSalmonella bacterium.
 21. The process of claim 14, wherein the carriermolecule is CRM₁₉₇.
 22. The process of claim 14, wherein the additionallinker is adipic acid dihydrazide (ADH).
 23. The process of claim 14,wherein the polysaccharide comprises an O-antigen and a core from alipopolysaccharide from a Salmonella bacterium, wherein the carriermolecule is CRM₁₉₇, wherein the additional linker is adipic aciddihydrazide (ADH), wherein the linker is adipic acidN-hydroxysuccinimide diester (SIDEA) and wherein the additional linkeris coupled using a carbonyl group at the reducing terminus of thepolysaccharide.